PROJECT SUMMARY/ABSTRACT Despite decades of study, how proteins fold in cells remains poorly understood. Protein folding and misfolding underlies the pathogenesis of diseases ranging from cancer to neurodegeneration. Much of what we do know about protein folding has been gathered from in vitro experiments, which do not fully model the complex intracellular environment including chaperones, membranes, and other biomolecules. Furthermore, our current knowledge of folding, both in vitro and in vivo, primarily relies on low-throughput, single-protein experiments. While providing great detail, these methods cannot simultaneously test how differential folding and misfolding across the proteome impacts disease physiology. Over 20% of human proteins contain disulfide bonds, and formation of these bonds typically represents the rate-limiting step in achieving the native fold under oxidizing conditions. Therefore, monitoring the kinetics of native disulfide bond formation can provide a proxy for successful protein folding (Mamathambika and Bardwell, 2008). Our group has pioneered the use of targeted mass spectrometry to monitor cellular protein synthesis. Here, we propose an entirely new approach to monitor oxidative protein folding across hundreds of proteins simultaneously: using targeted, quantitative mass spectrometry to monitor the kinetics of native disulfide bond formation in vivo. In my group we specifically focus on the study of multiple myeloma, a hematologic malignancy of plasma cells with no known cure. This disease is fundamentally a disorder of aberrant protein homeostasis: it is thought that unregulated production of immunoglobulin leads to many of the known clinical sequelae, while inducing apoptosis by increasing unfolded protein stress is a first-line therapeutic strategy. Here, we will first develop biochemical and proteomic tools to monitor native disulfide bond formation in nascently synthesized proteins within the endoplasmic reticulum. We will then use these tools, in combination with tuning of immunoglobulin protein synthesis through CRISPR inhibition and activation, to test the clinically-relevant hypothesis that myeloma cells are exquisitely sensitive to proteasome inhibition due to increased unfolded protein stress. Finally, we will test the effects of modulation of oxidative folding chaperones on simultaneous folding kinetics across many classes of myeloma-relevant secreted and extracellular proteins. We anticipate that these experimental approaches will provide a significant advance toward our understanding of global protein folding in vivo, thereby addressing a major gap in our knowledge of this central biological process. Furthermore, our results here will provide a breakthrough toward the study of a broad range of intracellular protein dynamics that are inaccessible with other methods.